a) Field of the Invention
The present invention relates to radiation sensitive liposomes and the use of these liposomes as carriers for therapeutic and diagnostic agents. The invention further relates to methods of producing the radiation sensitive liposomes and to methods of diagnosing and treating cancer and other conditions and diseases.
b) Description of Related Art
Liposomes are microscopic vesicles consisting of concentric lipid bilayers. Structurally, liposomes range in size and shape from long tubes to spheres, with dimensions from a few hundred Angstroms to fractions of a millimeter. Regardless of the overall shape, the bilayers are generally organized as closed concentric lamellae, with an aqueous layer separating each lamella from its neighbor. Vesicle size normally falls in a range of between about 20 and about 30,000 nm in diameter. The liquid film between lamellae is usually between about 3 and 10 nm. A variety of methods for preparing liposomes have been described in the literature. For specific reviews and information on liposome formulations, reference is made to reviews by Pagano and Weinstein (Ann. Rev. Biophys. Bioeng. 1978, 7:435–68) and Szoka and Papahadjopoulos (Ann. Rev. Biophys. Bioeng. 1980, 9:467–508) and additionally to U.S. Pat. Nos. 4,229,360; 4,224,179; 4,241,046; 4,078,052; and 4,235,871, the disclosures of which are incorporated by reference herein.
Biological cell membranes exploit the amphiphilic nature of lipids to create anatomical boundaries, e.g., the plasma membrane and the mitochrondrial membrane. During the early 1960s researchers demonstrated that certain classes of lipids, especially glycerophospholipids, could be used to form protein-free model membranes. They developed methods for the preparation of supported bilayer lipid membranes (BLM) and discovered that dried thin films of phospholipids spontaneously hydrate to yield self-supported closed bilayer assemblies of several thousand lipid molecules, i.e., liposomes. The lipid bilayer in each model membrane is a two-dimensional fluid composed of lipids with their hydrophilic head groups exposed to water and their hydrophobic tails aggregated to exclude water. The bilayer structure is highly ordered, yet dynamic due to the rapid lateral motion of the lipids within the plane of each half of the bilayer.
Typically, liposomes can be divided into three categories based on their overall size and the nature of the lamellar structure. The three classifications, as developed by the New York Academy Sciences Meeting, “Liposomes and Their Use in Biology and Medicine,” of December, 1977, are multi-lamellar vesicles (MLV's), small uni-lamellar vesicles (SUV's) and large uni-lamellar vesicles (LUV's). SUV's range in diameter from approximately 20 to 50 nm and consist of a single lipid bilayer surrounding an aqueous compartment. Unilamellar vesicles can also be prepared in sizes from about 50 nm to 600 nm in diameter. While unilamellar vesicles are of fairly uniform size, MLV's vary greatly in size up to 10,000 nm, or thereabouts, are multi-compartmental and contain more than one bilayer. LUV liposomes are so named because of their large diameters which range from about 600 nm to 30,000 nm; they can contain more than one bilayer.
Liposomes may be prepared by a number of methods, not all of which can produce the three different types of liposomes. For example, ultrasonic dispersion by means of immersing a metal probe directly into a suspension of MLV's is a common way for preparing SUV's. Preparing liposomes of the MLV class usually involves dissolving the lipids in an appropriate organic solvent and then removing the solvent under a gas or air stream. This leaves behind a thin film of dry lipid on the surface of the container. An aqueous solution is then introduced into the container with shaking in order to free lipid material from the sides of the container. This process disperses the lipid, causing it to form into lipid aggregates or liposomes. Liposomes of the LUV variety may be made by slow hydration of a thin layer of lipid with distilled water or an aqueous solution. Alternatively, liposomes may be prepared by lyophilization. This process comprises drying a solution of lipids to a film under a stream of nitrogen. This film is then dissolved in a volatile, freezable, organic solvent, e.g., cyclohexane or t-butanol, frozen, and placed on a lyophilization apparatus to remove the solvent. To prepare a pharmaceutical formulation containing a water-soluble drug, an aqueous solution of the drug is added to the lyophilized lipids, whereupon liposomes are formed.
Lipophilic drugs may be incorporated into the bilayer by dissolving them with the lipid in the organic phase and then removing the organic phase. Hydration with the aqueous phase will result in the incorporation of the lipophilic drug into the liposomal bilayer structure. This applies both to lyophilization and thin film methods. The ratio of drug to lipid may be up to about 20% by weight, preferably from about 0.001% to about 0.1%. The encapsulation characteristics and biocompatibility of liposomes make them ideal carriers for therapeutic agents. Research efforts have been devoted to the development of liposomes for the delivery of drugs in the body. Successful in vitro studies have led to clinical trials of liposome-encapsulated amphotericin B, anthracyclines, and other drugs. Suitably designed liposomes can extend the circulation time and target the drug to particular tissues of the body (Allen, T. M., Liposome Res. 1992, 2:289–305; Allen, T. M., Trends Pharm. Sci. 1994, 15:215–220; Blume et al., Biochim. Biophys. Acta 1993, 1149:180–184; Klibanov et al., J. Liposome Res. 1992, 2:321–334; Lasic et al., D. Science 1995, 267:1275–1276; Lasic et al., Stealth Liposomes, CRC Press: Boca Raton, Fla., 1995). The delivery of liposomes to the desired sites depends in part on long circulation times in the body, which can only be accomplished by reducing the uptake of liposomes by the mononuclear phagocytic system (MPS). In recent years several means have been described to sterically stabilize liposomes in order to increase their period of circulation (Lasic et al., Stealth Liposomes, supra; Woodle et al., Biochim. Biophys. Acta 1992, 1113:171–199). A frequently used method is the attachment of poly(ethylene glycol) (PEG) to some of the lipids in the liposome. This is usually accomplished by the chemical reaction of PEG or its derivatives with the amino function of phosphatidylethanolamines (PE), e.g., methyl-PEG coupled to PE via a carbamyl linkage (Allen et al., Biochim. Biophys. Acta 1991, 1066:29–37); activation of methoxy-PEG with cyanuric acid (Klibanov et al, FEBS Lett. 1990, 268:235–243; Mori et al., FEBS Lett. 1991, 284:263–271); and conjugation of PEG to PE with succinimidyl succinate (Klibanov et al., FEBS Lett., supra; Mori et al., FEBS Lett., supra; Woodle et al. Proceed. Intern. Symp. Control. Rel. Bioact. Mater 1990, 17:77). PEG-modified PE may be incorporated into liposomes by including it with the other lipids during the formation of the liposomes. Alternatively, monomethoxy-PEG has been coupled to the outer surface of preformed liposomes, which contains some fraction of PE (Senior et al., Biochim. Biophys. Acta 1991, 1062:77–82). Regardless of the means of PEG incorporation, the inclusion of PEG ranging in size from about 1000 to 5000 daltons results in liposomes with an order of magnitude or greater increase in circulation time in the body. The useful mole fraction of PEG/PE depends on the polymer chain length. Thus 5 mole percent of PEG1900, wherein the 1900 indicates the number average molecular weight of the PEG, was effective in achieving increased circulation time, whereas 15 mole percent of PEG750 was necessary to achieve a comparable stabilization (Allen et al., Biochim. Biophys. Acta, supra). These results suggest that a minimum necessary surface coverage of the liposome is achieved at a lower mole fraction of the longer polymer. Many studies have utilized 100 nm diameter LUV composed of various phosphatidylcholines (PC) and PEG-PE or PC/cholesterol/PEG-PE in different molar ratios (Woodle et al., Biochim. Biophys. Acta, supra). The incorporation of PEG-PE into LUV composed of PC/PE is effective in increasing their circulation time. Although larger liposomes generally have a shorter circulation time than 100 nm LUV, it has been shown that the inclusion of PEG-PE in liposomes as large as 250 nm increases their circulation time (Woodle et al., Biochim. Biophys. Acta, supra). Steric stabilization of liposomes by PEG-PE is reported to be effective for liposomes in both the solid-analogous and liquid-analogous phases (Allen et al., Liposome Res., supra).
In addition to extended circulation times, the successful delivery of liposomes to specific tissue sites requires the liposomes to enter the interstitium. Tumors represent a specific tissue site of considerable therapeutic interest; several research groups have reported the increased localization of sterically stabilized liposomes (PEG-liposomes) at tumor sites. The increased permeability of the vasculature at tumor sites (due to angiogenic factors secreted by tumors) allows liposomes to escape the capillaries to reach the tumor interstitial space. Sterically stabilized liposomes are more likely to accumulate at these sites because of their sustained concentration in the blood. Furthermore, it is known that the hydrophilic surface polymer may facilitate the transit from the capillaries to the tumor site. Reports of passive targeting of PEG-liposomes to tumors, including murine colon carcinomas, murine lymphomas, murine mammary carcinomas, human squamous cell lung carcinomas in SCID mice are known in the art. Specific targeting via antibodies coupled to liposomes has been observed as well. Antibody (mAb) conjugated sterically stabilized liposomes are known to localize at squamous cell carcinomas of the lung in mice and effectively deliver doxorubicin to these sites. Although the coupling of mAbs to conventional liposomes appears to increase their rate of clearance from the blood stream, the mAb conjugated PEG-liposomes remain in circulation long enough to accumulate at their target cells.
In order for the liposomes to reach the target site without significant loss of their contents, passive leakage must be slow relative to the time required for liposomes to circulate and escape the vasculature. However, it has been shown that once sterically stabilized liposomes have accumulated at tumor sites the slow passive leakage of encapsulated chemotherapeutics, e.g., doxorubicin, can significantly affect the cells at that site. It would be desirable to stimulate enhanced release of the encapsulated agent(s) from the liposomes once the liposomes are at the target site. Ideally, such a stimulus would be spatially and temporally selective, in a manner analogous to photodynamic therapy. In photodynamic therapy, certain porphyrins and other photosensitizers are administered systemically, absorbed by cells, and upon exposure to visible light focused at the target site. Hence, the photodynamic effect results in the localized destruction of the target cells. This effect has proven useful for the treatment of cancer cells in areas of the body that are accessible to coherent light via fiber optics. In principle, the successful use of light (or other forms of radiation) to treat disease can be broadened to include a wide variety of therapeutic agents, particularly, if light is used to release the agent.
Several strategies have been employed to design photosensitive liposomes. These include the photochemical modification of individual lipids in the bilayer, i.e., lipid photochemistry; the photo induced change in the association of polyelectrolytes with liposomes; and the photoinitiated polymerization of some or all of the lipids in the liposome, i.e., photopolymerization. A characteristic of photopolymerization processes is their multiplicative nature, which generally results in a greater perturbation of the bilayer membrane for equivalent light exposures. An extensive review of methods to photochemically reorganize lipid bilayers has been published (O'Brien et al., Bioorganic Photochemistry 1993, 2:111–167).
The photopolymerization of selected lipids in a multicomponent membrane can alter the lateral distribution of lipids within the bilayer to form domains enriched in polymerized lipids (Armitage et al., Adv. Polym. Sci. 1996, 126:53–85). It is known that processes that cause the phase separation of PE and other lipids can trigger lamellar to nonlamellar phase transition(s). The polymerization of two (or multi) component lipid bilayers, with one polymerizable and other nonpolymerizable component(s), can cause lipid domain formation. The polymerizable lipids form covalently linked domains as the reaction proceeds, which in turn produces domains of the nonpolymerizable component(s). Recently, O'Brien and coworkers showed that if the nonpolymerizable component prefers a nonlamellar rather than a lamellar structure the membrane will be destabilized (Lamparski et al., Biochemistry 1992, 31:685–694; Bennett et al., Biochemistry 1995, 34:3102–3113). Phosphatidylethanolamines (PE) are of particular interest because they form nonbilayer structures under physiological conditions. Two-component liposomes of a polymerizable PC and a PE form stable liposomes prior to polymerization, but are destabilized by photopolymerization of a bis-SorbPC which contains a photosensitive sorbyl moiety at the terminal end of each acyl chain. Consequently, the photopolymerization of properly designed lipid bilayers can initiate the localized destabilization of the bilayer, which is observed either as the leakage of encapsulated reagents (Lamparski et al., supra) or the fusion of bilayer liposomes (Bennett et al., supra).
These original studies utilized the native photosensitivity of the bis-SorbPC due to its chromophore at 260 nm. In principle, liposome destabilization can be achieved by the polymerization of a host of reactive lipids that are known in the literature. The polymerization of supramolecular assemblies of amphiphiles was first demonstrated in monolayers, vesicles, and extended bilayers. The introduction of synthetic double tail amphiphiles coupled with the successful demonstration of polymerization of fatty acid monolayers led directly to the description of a cationic ammonium salt with a methacrylate at the end of one hydrocarbon chain in the early 1980s. Reports of the syntheses and polymerization of lipid diacetylenes in bilayers followed. Further, syntheses and polymerization of dienoyl lipids, additional methacryloyl lipids, other lipid diacetylenes, and vinyl lipids appeared as well (Ringsdorf et a., J. Angew. Chem. Int. Ed. Engl. 1988, 27:113–158; O'Brien et al., Encylopedia Polym. Sci. Engr. 1989, 17:108–135; O'Brien et al., Acc. Chem. Res 1998, 31:861–868). The polymerization of these reactive lipids in a self-organized array of several thousand lipid molecules into a structure that contains several polymer chains is termed the formation of polymerized liposomes.
A major strategy for the formation of polymerized bilayers and other supramolecular assemblies is the preparation of polymerizable lipid monomers, the formation of the lipid assembly such as bilayer membranes from the monomer, and the subsequent chain polymerization of the monomers in the assembly. Polymerizable lipids have been prepared by introduction of the reactive group into different regions of the lipid molecule. A schematic representation of these types of polymerizable lipids is shown in FIG. 1. As shown in FIG. 1, polymerization strategies A and B have no direct influence on the membrane-water interface. The mobility of the lipid chains is significantly decreased by polymerization in these systems. In contrast methods C and D alter the membrane-water interface, but have less effect on the hydrophobic interior of the membrane. These examples with only one reactive group per lipid form linear polymer chains in supramolecular assemblies. The presence of a second polymerizable group per molecule (not shown) allows crosslinking of the polymer chains.
A host of reactive moieties have been utilized to modify the above lipids to make them polymerizable. These groups include diacetylene, acryloyl, methacryloyl, itaconyl, dienoyl, sorbyl, muconyl, styryl, vinyl, thiol (or lipoyl), and chain terminal isocyanates. Systematic studies of the relationship between polymer chain length, i.e., degree of polymerization (Xn), and the molar ratio of monomer to initiator revealed that Xn was proportional to [M]/[I]. Moreover, these studies showed that the relative reactivity of monomers in bilayer membranes is similar to values obtained from the multitude of solution polymerization studies. Consequently, an acryloyl lipid monomer in a bilayer is four to five times more reactive than a diene containing lipid monomer.
Polymerization of lipids in bilayer membranes can be caused by various methods, including photo, thermal, and redox initiation. Diacetylenic, butadienic, vinylic, acryloylic, methacryloylic, and thiolic units have been used as polymerizable units in acyl chains. However, while the use of ionizing radiation to initiate chain polymerizations is known in the art, less is known about the effects of ionizing radiation in order to stabilize lipid bilayer membranes composed of polymerizable lipids. Akama et al. report the stabilization of liposomes by the polymerization of polymerizable phospholipids contained in the membrane. More specifically, they report stabilization of liposomes as a result of polymerization by hydroxyl radicals generated by gamma-irradiation. The design of polymerizable phospholipids is important for obtaining a stabilizable liposome by polymerization (Akama et al., J. Mater. Chemistry 2000, 10:1047–1059). However, while the use of ionizing radiation to stabilize lipid bilayer membranes composed of polymerizable lipids is known, there are no reports about the use of ionizing radiation for the purpose of liposome destabilization.
The methods described above relating to radiant energy initiated polymerization of the lipid bilayer rely on ultraviolet light. The potential utility of polymerizable liposomes for drug delivery, diagnostics, and reagent release is limited if only ultraviolet light can be used for initiation of polymerization. UV light can only be used where the target tissue is superficially accessible to the light source. Liposomes that exist at deeper tissue levels would not be accessible to UV light and liposome-encapsulated or associated diagnostic or therapeutic agents could therefore not be released. Hence, a better system is required to achieve destabilization of liposomes. All publications, patents, and other reference materials referred to herein are incorporated herein by reference.